When cultured in gene-manipulated animal cells, monoclonal antibodies are secreted into a medium and present in very low concentrations while being mixed with various proteins secreted from the cells and those proteins in the medium. Therefore, the removal of impurities other than the desired monoclonal antibodies is an important step in antibody production. Affinity chromatography using protein A, which is an antibody affinity ligand capable of selectively recovering monoclonal antibodies from a medium, is mainly used for the separation and purification of monoclonal antibodies.
After antibody purification, cleaning-in-place (CIP) is performed to remove various contaminants such as nucleic acids, lipids, proteins, and microorganisms remaining in the resin. In general, NaOH is the most widely used material among resin cleaners. However, since protein-based refining resins are vulnerable to the alkali, there is a limitation to use NaOH. Protein A is washed with NaOH because it is relatively stable even under alkaline conditions. However, in order to increase the cleaning efficiency more, protein improvement studies have been carried out to develop protein A that is stable even under alkaline conditions.
Protein A is the cell surface protein of Staphylococcus aureus and is composed of five highly homologous domains (E, D, A, B, and C domains) which have a structure consisting of three semi-parallel helices and two loops located between with about 58 amino acid residues. Based on the streptococcal strain-derived albumin-biding domain (streptococcal albumin-binding domain), Gulich replaced all Asn residues with other amino acids based on the fact that Asn residues were sensitive to the alkali, resulting in obtaining a protein having high thermal stability and stability against 0.5 N NaOH (Gulich et. al., J Biotechnol, 28(2), 169-178 (2000)). Linhult developed a type of Protein A which retains its structural stability toward repeated alkaline treatments using bypass mutagenesis approach. More specifically, F30 of the Z domain (G29A variant of the B domain) exists at the third helix position and thus does not interfere with antibody binding, while being a residue that affects the structural stability. In fact, when F30 was replaced with Ala, its affinity to IgG was similar to wild type, but its structural stability was reduced, resulting in weakening its to resistance to the alkali its. When Z (F30A) was used as a template and Asn residues were replaced with residues other than Asn existing at the same position in other domains (E, D, A, and C domains), that is, when it was changed to N23T, N28A, N6A and N11S, it was confirmed that its stability increased under the alkaline condition. However, considering that their alkali tolerance is lower than that of the wild type, in the case of N21A, N43E and N52A, in addition to simply replacing of Asn with other residues, it is also important to know what type of residues have been replaced (Linhult et. al., Proteins, 55(2), 407-416 (2004)).
Korean Patent No. 10-1307651 discloses a mutant immunoglobulin-binding protein in which N3A/N23T or N3A/N6DN23T in the B domain or Z domain has occurred, which has an increased chemical stability at alkaline pHs compared to its parent molecule. U.S. Pat. No. 9,051,375 describes mutant sequences with an increased resistance to the alkali by substituting with histidine, serine, aspartic acid or threonine at three positions of N3, N6 and N23 in the Z domain, respectively.
Further, U.S. Patent Application Publication No. US20100048876 describes a chromatographic matrix comprising a C domain with the 3rd to 6th residues from the N terminus removed and a wild-type C domain, which has an increased stability forward the alkali. Korean Patent No. 10-1464040 discloses affinity chromatography ligands and matrices comprising two to five C, B or Z domains with three or four consecutive amino acid deletions from the first amino acid of the N-terminus, and this matrix is characterized by low ligand degradation under alkaline conditions compared to wild type ligands.
As described above, as individual Protein A ligands, the increase in the alkali tolerance was confirmed by substituting several specific amino acids for asparagine residue or eliminating the N terminal residues. However, such an effect is limited, considering that substitution residues affect alkali tolerance.